This project is concerned with the mechanism of calcium-evoked neurotransmitter release in the central nervous system. This is the basic mechanism of perception, thought and responsiveness. When considering the nature of this mechanism, attention is shifting from protein phosphorylation to the synaptic membrane and its underlying cytosketetal elements which are hypothesized to rearrange under the influence of a calcium influx, thereby permitting synaptic vesicle fusion with the presynaptic membrane. Two important proteins which participate in membrane-cytoskeletal interactions are spectrin and tropomyosin. It has been observed that both of these proteins bind calcium specifically with high affinity. Experiments are proposed to examine this calcium binding in more detail. Flow dialysis experiments will be used to determine the association constants and numbers of binding sites of divalent cations. Stopped flow fluorescence studies will provide the divalent cation on and off rates. The proteins will be cleaved and the fragments will be analyzed for calcium binding to localize the binding sites. Other studies will examine the effects of divalent cation binding on the interactions of these proteins with other proteins in cytoskeletal complexes. Because several diseases have been associated with alterations of neurotransmitter release, solution of the questions to be examined by this project should have broad implications for the improvement of mental health.